Team:Bielefeld-CeBiTec/Project/Mutation

Mutation

Power through diversity

Overview

One of the elementary requirements of every evolutionary process is variability, which can be achieved by mutagenesis. The alteration of existing genotypes is the only way to discover new features and abilities of proteins and organisms. The aim of our mutagenesis system is to create a diversity of binding proteins during the course of fermentation to gain as much different Evobodies as possible. Building on the library approach and leading to the selection system, the mutagenesis represents the central part of our project. After transformation of the library and an initial selection round the mutagenesis has to take over. That is why we are using a system that is capable of in vivo DNA alteration. Two different approaches were applied to reach this aim.

The mutagenesis plasmid is rather unspecific and leads to a general raise of mutations in the DNA of host organisms. This system was used to get a manifold increase of base pair exchanges and a strong repression of bacterial DNA repair mechanisms. Six different proteins build this mutation system.

A more specific approach is the two plasmid mutagenesis system (Camps, 2003). It is based on a modified DNA polymerase I, which replicates the first part of plasmids belonging to the ColE1-family, like pSB1C3.

In the following, we will elaborate on why and how we implemented these two systems in detail in our project.